The assembly and function of a variety of biological molecular machines is key to the structure and function of the cell. Because of their complexity, mechanistic details of the assembly of these machines is often hidden in ensemble experiments. In this project, we will develop and use multicolor single molecule FRET methods to study an important molecular machine, the 30S subunit of the bacterial ribosome. 3- and 4-color FRET methods will provide information about coupling of conformational changes and binding, as well as conformational changes in well-separated parts of the assembling subunit. We will use these single-molecule methods to probe the assembly process at multiple levels, by studying a series of ribosomal constructs of different size and complexity. We will first probe conformational changes in small protein-binding modules from the platform domain. We will also study global changes in the isolated head and the entire subunit as a function of protein binding as well as at key intermediates along the assembly pathway. We expect that these studies will provide new methods and insights into the assembly that will be useful to the general biological community, and also might help understand how to design improved antibiotics. [unreadable] [unreadable]